Abstract The goal of this grant is to study how microtubules, actin filaments, and motor proteins generate polarity in Drosophila oocytes. Oocyte polarity is critical for early embryonic development, and polarity determinants are conserved between Drosophila and humans, allowing us to study polarity determination using a genetic system amenable to high-resolution imaging. We will use live imaging to study molecular mechanisms of transport and posterior oocyte anchoring of an evolutionary conserved polarity determinant Staufen. We will test a hypothesis that Staufen is transported in the nurse cells of Drosophila ovaries and between individual cells through the ring canals in the form attached to microtubules, and that these microtubules are moved in the cytoplasm and through the ring canals by molecular motors. We will identify these motors and find the proteins that attach Staufen to microtubules. Our lab has discovered that conventional kinesin has a new and conserved function of sliding microtubules against each other and generated new genetic tools that allow us to microtubule-sliding and cargo-transporting functions of kinesin. Using these tools, we will determine the role of microtubule-microtubule sliding in kinesin localization. We will explore the role of the cortical network of actin filaments in the localization of Staufen and will test the hypothesis that competition between cortical anchoring to actin and transport by or along microtubules defines the final asymmetrical distribution of Staufen. We will identify proteins that link Staufen with the posterior cortex in the oocyte. This work will advance our knowledge of fundamental transport processes that define cell polarity, and early embryonic development.